The present invention relates generally to sodium channel proteins and more particularly to mammalian cardiac sodium channel proteins, to DNA sequences encoding sodium channel proteins, to the polypeptide products of recombinant expression of these DNA sequences, to peptides whose sequences are based on amino acid sequences deduced from these DNA sequences, to antibodies specific for such proteins and peptides, and to procedures for detection and quantitation of such proteins and nucleic acids related thereto, as well as procedures relating to the development of anti-arrhythmic and cardiotonic drugs based on interaction with such proteins.
Cardiac arrhythmias are responsible for 15-20% of the deaths in the United States; there are approximately 2,000,000 congestive heart failure patients in this country, with about 250,000 new cases each year. The cardiac sodium channel protein (also referred to as cardiac sodium channel) is intimately involved in most lethal arrhythmias and is the site of action of most clinically effective anti-arrhythmic drugs such as lidocaine, tocainide, quinidine, etc; and cardiotonic drugs that enhance cardiac contractility, including a new class of cardiotonic agents of which one (DP I 201-106) is presently in clinical trials.
Sodium channels are transmembrane proteins responsible for the early sodium permeability increase underlying the initial depolarization of the action potential in many excitable cells, such as muscle, nerve, and cardiac cells. Specifically, cardiac sodium channels are responsible for the excitation and conduction of the action potential (AP) in myocardial cells. Fozzard, H.A., et al., Circ. Res., 56:475-485 (1985).
Cardiac sodium channel involvement in the generation of cardiac action potentials makes the channel a crucial site where anti-arrhythmic and cardiotonic agents act. The sodium channel is the most thoroughly characterized of the voltage-gated channels at this time. Six distinct neurotoxin or drug receptor sites have been characterized on the sodium channel, associated with channel pore or gating structures. Catterall, W.A., Ann. Rev. Biochem. 55:953-985 (1986); Catterall, W.A., ISI Atlas of Science: Pharmacology 190-195 (1988). Two functionally distinct populations of sodium channel subtypes have long been hypothesized to account for the physiological observation in certain mammalian cell preparations of two components of sodium current which have differing relative sensitivity to the neurotoxins, tetrodotoxin (TTX) and saxitoxin (STX). "TTX-sensitive" (TTX-S) sodium channels in mammalian nerve and in skeletal muscle are blocked by nanomolar concentrations of STX and TTX. "TTX-resistant" (TTX-R), or "TTX-insensitive" sodium channels, first observed in mammalian denervated skeletal muscle and in heart, have sodium currents and STX/TTX receptor sites which are blocked only by 2-3 orders of magnitude higher concentrations of TTX, i.e., in the 1-10 .mu.M concentration range. TTX-resistant sodium channels have subsequently been found in widespread distribution in other immature mammalian nerve and in skeletal muscle cells lacking mature innervation. Electrophysiological and pharmacological studies have made it clear that TTX-resistant mammalian cardiac sodium channels have specialized properties distinct from TTX-sensitive sodium channels in nerve and skeletal muscle. The mechanisms by which drugs and neurotoxin agonists and antagonists act at the sodium channel and the development of general rules for how drugs interact with other ion channels having extensive homologies can be more readily studied once the actual structure of the sodium channel isoforms is more clearly understood. As used herein, the term "isoform" is used to mean distinct but closely related sodium channel proteins, which show strong homology in amino acid sequence and function and the term "subtype" is used to mean different major forms of sodium channels as identified by selective pharmacological agents showing different channel affinities.
Biochemical studies have shown [Catterall, W.A., Ann. Rev. Biochem., 55:953-985 (1986)] that a large, approximately 260-kDa, glycoprotein .alpha.-subunit is common to purified channel preparations from Electrophorus electricus (electric eel) electric organ, rat brain, rat skeletal muscle, and chick heart muscle. Rat brain contains, in addition, two smaller polypeptide subunits (.beta.-1 and .beta.-2) of molecular weight 36-kDa and 33-kDa respectively. The .alpha.- and .beta.-2 subunits are covalently attached by disulfide bonds. Rat skeletal muscle also contains at least one .beta.-subunit. The .alpha.-subunit alone appears to be responsible for specifying many of the key sodium channel functions. Noda, M., et al., Nature, 322:826-828 (1986); Agnew, W.S., Nature, 322:770-771 (1986); Goldin, A.L., et al., Proc. Nat'l. Acad. Sci. USA, 83:7503-7507 (1986). However, the .beta.-subunits may modulate sodium channel functional properties as well. Krafte, D.S., et al., J. Neurosci, 8:(in press) (1988). Other studies [Catterall, W.A., et al., Molec. Pharmacol., 20:533-542 (1981); Frelin, C., et al., Pflugers Arch., 402:121-128 (1984); Rogart, R.B., Ann. New York Acad. Sci., 479:402-430 (1986); Moczydlowski, E., et al., Proc. Nat'l. Acad. Sci. USA, 83:5321-5325 (1986)] have revealed the existence of multiple closely related isoforms of the sodium channel found in different animal species, in different tissues within the same species, and even in the same tissue.
Cloning studies of cDNAs encoding the sodium channel large .alpha.-subunit from eel electroplax [Noda, M., et al., Nature 312:5990 (1984)], rat brain [Noda, M., et al., Nature 320:188-192 (1986)], and Drosophila [Salkoff, L., et al., Trends in Neuroscience (1987); Salkoff, L., et al., Science 237:744-749 (1987)] have demonstrated that: 1) the sequence of the .alpha.-subunit consists of four repeated, highly homologous hydrophobic domains (each of which contains six transmembrane segments of S1-S6) separated by hydrophilic, nonrepeated intervening sequences; 2) considerable homology exists among the sequences from different species, with the greatest conservation existing among the four internally homologous domains; 3) the S4 segment of each homologous domain is positively charged, with four to eight lysine or arginine residues at every third position, which may be involved in channel gating [Greenblatt, R.E., et al., FEBS 193:125-134 (1985); Guy., R.H., et al., Proc. Natl. Acad. sci. USA 83:508-512 (1986); Noda, M., et al., Nature 312:5990 (1984)]; 4) in rat brain [Noda, M., et al., Nature 320:188-192 (1986); Kayano, T., et al., FEBS Letters 228:187-194 (1988)], three homologous brain mRNA sequences (designated as types I, II, and III) encode distinct sodium channel isoforms in the same tissue; and 5) expression of mRNA injected into oocytes, coding for the .alpha.-subunit alone of the rat brain I, II, or III sodium channels, was sufficient to produce a functional voltage-activated sodium channel [Noda, M., et al., Nature 322:826-828 (1986); Suzuki, H., et al., FEBS Letters 228:195-200 (1988); Agnew, W.S., Nature 322:770-771 (1986); Goldin, A.L., et al., Proc. Natl. Acad. Sci. USA 83:7503-7507 (1986)] exhibiting many of the key properties of the native channel, including appropriate kinetics, voltage-sensitivity, ion selectivity, and sensitivity to the neurotoxin TTX. Different groups have found .beta.-subunits important to varying extents to sodium channel function, making their role somewhat controversial. Catterall, W.A., Ann. Rev. Biochem. 55:953-985 (1986); Agnew, W.S., Nature 322:770-771 (1986); Goldin, A.L., et al., Proc. Natl. Acad. Sci. USA 83:7503-7507 (1986); Messner, D.J., et al., J. Biol. Chem. 261:14882 (1986); Auld, V.J., et al., Neuron (in press) (1988); and Stuhmer, W., et al., Eur. Biophys. J. 14:131-138 (1987).
The detection of three separate cDNA clones has led to the identification of three structurally distinct sodium channel isoforms in rat brain. Noda, M., et al., Nature 320:188-192 (1986). Two further distinct isoforms have been detected in rat skeletal muscle [Barchi, R.L., Probing the Molecular Architecture of the Voltage-Dependent Sodium Channel in "The Molecular Biology of Receptors, Pumps, and Channels: Pharmacological Targets," ASPET Meeting Abstracts; Abstract No., Aug. 1988]. The molecular relationship of these isoforms found in rat brain and in rat skeletal muscle to the sodium channel isoforms found in rat heart remains unknown.
Attempts have been made to clarify the functional relationship of the three rat brain isoforms. When rat brain mRNA species were injected into Xenopus oocytes, it was found that II and III both induced similar sodium currents [Noda, M., et al., Nature, 322:826-828 (1986); Suzuki, H., et al., FEBS Letters, 228:195-200 (1988)]; and that these currents were also grossly similar to those produced upon injection of rat brain poly(A+) mRNA. Stuhmer, W., et al., Eur. Biophys. J., 14:131-138 (1987). However, injection of rat brain I sodium channel mRNA into oocytes induced only small currents. Further, recent studies have detected characteristics of the sodium current which differ when induced by sodium channel-specific mRNAs than when induced by those from total rat brain poly (A+) mRNA. Mandel, G., et al., Proc. Nat'l. Acad. Sci., 85:924-928 (1988); Auld, V.J., et al., Neuron, 1:449-461 (1988); Krafte, D.S., et al., J. Neurosci., 8:(in press) (1988). These conflicting results make it difficult to determine the functional relationship between the three rat brain sodium channel mRNA species and the functional properties of the sodium channel isoforms they encode. Furthermore, biophysical descriptions of nerve membrane sodium permeability do not provide a clearcut role for as many as three sodium channel isoforms. For instance, voltage-clamp studies of the sodium current in mammalian nerve cells have most frequently been interpreted in terms of only a single population of sodium channels. Aldrich, R., et al., J. Neurosci., 7:418-431 (1987).
It remains unclear 1) which structural domains of the human TTX-resistant cardiac sodium channel account for their specialized functional properties; 2) what the role of multiple cardiac sodium channel isoforms in cardiac action potential excitation and conduction is; and 3) the nature of the interaction of pharmacological agents with the small sequence domains which form the receptors. Further, it remains unclear as to how these TTX-sensitive and TTX-resistant sodium channel isoforms may arise.
Two apparently different brain and cardiac sodium channel isoforms may represent post-translational modifications of a single polypeptide molecule; the same .alpha.-subunit may be present in both cardiac and nerve sodium channel isoforms, and differences may arise from the presence of other small .beta.-subunits making up the channel protein; and/or distinct .alpha.-subunits may account for the differences between the TTX-sensitive and TTX-resistant isoforms of the sodium channel. These distinct .alpha.-subunits may be encoded by distinct gene sequences which arise from a family of closely related genes which are differentially expressed in various tissues, or from alternative splicing of a single gene.
There thus continues to exist a need in the art for information concerning the ways in which these TTX-sensitive and TTX-resistant sodium channel isoforms arise, as well as specific information concerning the primary structural conformation of cardiac sodium channel protein and concerning other sodium channel proteins such as might be provided by knowledge of human, rat, and other mammalian DNA sequences encoding the same.
Availability of such DNA sequences would make possible the application of recombinant methods to the large scale production of the proteins in procaryotic and/or eukaryotic host cells, as well as DNA-DNA, DNA-RNA, and RNA-RNA, hybridization procedures for the detection, quantification and/or isolation of nucleic acids associated with these proteins. Possession of cardiac sodium channel and related sodium channel proteins and/or knowledge of the amino acid sequences of the same would make possible, in turn, the development of monoclonal and polyclonal antibodies thereto (including antibodies to protein fragments or synthetic peptides modeled thereon) for the use in immunological methods for the detection and quantification of the proteins in fluid and tissue samples, as well as for tissue specific delivery of substances such as labels and therapeutic agents to cells expressing the proteins; as well as allowing for the development of new anti-arrhythmic and cardiotonic drugs.